DESCRIPTION: Delivery still remains as a barrier to achieving successful gene therapy. Administering gene delivery protocols in a manner that would allow better control over the expression pattern would enhance therapeutic outcomes. We have developed a delivery approach (gene electro transfer; GET) which utilizes pulsed electric fields that allows for controlled delivery. We have tested this approach as a means of delivering plasmids encoding immunostimulatory molecules. For immunotherapy, maintaining control over expression following plasmid delivery is critical to success as there is a fine balance between immunostimulation and immunosuppression. Manipulation of GET parameters can be used for controlled delivery of plasmid and will result in obtaining the appropriate transgene expression. The model system utilized to test this system is malignant melanoma which is a major health concern with no effective therapy for advanced disease. The incidence of melanoma continues to rise and it is estimated that there will be 76,690 new cases and 9,480 deaths in 2013. Melanoma is a good model for immunotherapy approaches as there is evidence demonstrating immune responsiveness including both innate and adaptive immunity. Recently, several new approaches have been tested as potential immunotherapies with some success. However, overall durable complete response rates (disease free survival) are low (<15%) and some of these therapies have significant adverse events documenting that there is still a need for more effective therapies. One potential new therapy is to deliver a plasmid encoding Interleukin-12 directly to the tumor to stimulate an immune response. The important criterion for success is administering IL-12 at the right dose and location. To address this, we have developed an effective means of delivering plasmid DNA utilizing GET. The hypothesis to be tested is: if appropriate delivery parameters are used to deliver plasmid IL-12 then a change in the tumor microenvironment will occur that will be associated with an appropriate therapeutic response. Therefore, it is critical to characterize the response and identify potential biomarkers that can signify proper delivery and expression. We also hypothesize that if an appropriate combination can be achieved then there will be an increased response at distant sites. The increased response rates together with boosting the immune response may lead to an effective therapy for metastatic melanoma due to a reduction of T-reg cells and enhanced activation of T-effector and memory cells. In this project, we will develop and test this approach in a mouse model and have the opportunity to determine how it correlates with samples obtained from an ongoing clinical trial. Thus, the work in this project is directly translatable. The following specific aim will be performed as part of this project. 1. Determine the influence expression profile has in inducing an effective anti-tumor response and determine if a specific pattern of response can be identified. 2. Evaluate expression patterns following delivery of plasmids encoding anti-PD1, anti-PD-L1 or anti-CTLA4. 3. Therapeutic efficacy of the approach in a mouse metastatic model.